The present invention discloses nucleotide sequences from the genome of Moraxella catarrhalis. These sequences may be used in various assays and in the development of diagnostic and therapeutic agents.
All animals coexist with an indigenous microflora. Beginning shortly after birth, the gastrointestinal tract, lungs, and other areas of the human body are colonized by different bacterial species. A large number of factors operate to maintain symbiotic, host-microbe balance. These include the physical barriers of skin and mucosal surfaces and both nonspecific and highly specific aspects of the immune system. When host-microbe balance becomes disturbed, infection may ensue. Virulence, the ability of a microbe to produce infection, is related to a variety of complex mechanisms of disease induction. Some organisms are highly virulent and cause clinical illness when they colonize most or all hosts. Alternatively, when host defenses are compromised, normally symbiotic microbes can induce serious, or even life-threatening, infections. Thus, infection is generally a consequence of the interaction between a relatively virulent microbe and a normal host or between a relatively less virulent microbe and a host with some degree of transient or permanent immunological impairment.
M. catarrhalis (Branhamella catarrhalis) is a large, aerobic, gram-negative diplococcus normally found among the bacterial flora of human upper airways. It is nonmotile and possesses fimbriae. Collonies are regularly friable and nonadherent and grow well on blood or chocolate agar. Unlike many other pathogenic bacteria, M. catarrhalis shows a high degree of homogeneity in its outer membrane proteins. This usually harmless parasite of the mucous membranes may behave as an opportunistic pathogen when microbe-host balance is perturbed. Following infection, host antibodies directed against one or more of the microbial outer-membrane proteins are detectable-in the serum.
M. catarrhalis is known to cause acute, localized infections such as otitis media, sinusitis, and bronchopulmonary infection and life-threatening, systemic diseases including endocarditis and meningitis. The presence of bacterial endotoxin and host histamine and chemotactic factors are major indicators of M. catarrhalis pathogenicity.
M. catarrhalis can be isolated from the upper respiratory tract of 50% of healthy school children and 7% of healthy adults. In children with otitis media, colonization increases to 86%, and it is the third most common bacterial isolate. It causes 10-15% of otitis media and sinusitis. Infections of the maxillary sinuses, middle ears, or bronchi may occur through contiguous spread of the microbes. M. catarrhalis causes a large proportion of lower respiratory tract infections in elderly patients with chronic obstructive pulmonary diseases and is exceeded only by Haemophilus influenzae and Streptococcus pneumoniae as a causative agent of acute purulent exacerbations of chronic bronchitis.
Pneumonia due to M. catarrhalis, like that of H. influenzae or S. pneumoniae, begins with aspiration of the bacteria. Failure or absence of appropriate host defense allows the bacteria to replicate and produce an inflammatory response in the alveoli. Because of mandatory immunosuppression, organ transplant recipients can develop moderate to severe M. catarrhalis pneumonia very rapidly. Bloodstream invasion is less characteristic of M. catarrhalis than pneumococcal infection, but nearly 50% of M. catarrhalis pneumonia patients die within 3 months of onset.
M. catarrhalis is treated with antibiotic agents including penicillin-clavulanic acid combinations, cephalosporins, tetracycline, erythromycin, chloramphenicol, trimethoprim-sulfamethoxazole, and quinolones. Over 85% of M. catarrhalis clinical isolates have been reported to be resistant to penicillin. Moreover, the microbe protects itself by binding to the first subcomponent of the complement system (Clq) which inactivates the C1 complex or by inactivating the terminal, lytic complement complex via a protein on the outer cell wall surface. Resistance is mediated by two closely related xcex2-lactamases, BRO-1, present in 90% of resistant isolates and BRO-2, present in 10%. These enzymes are active against penicillin, ampicillin, and amoxicillin, less active against cephalosporins, and bind avidly to clavulanic acid and sublactam. Tetracycline resistant strains are increasing in Europe and Asia and have been documented in the United States. Ampicillin, which had been universally effective in treating M. catarrhalis pneumonia, can no longer be used.
M. catarrhalis physiology and pathogenicity are reviewed in: Holt et al. (1994) Bergey""s Manual of Determinative Bacteriology, Williams and Wilkins, Baltimore Md.; Cullmann (1997) Med Klin 92(3):162-166; Isselbacher et al. (1994) Harrison""s Principles of Internal Medicine, McGraw-Hill, New York N.Y.; Murray (1995) Manual of Clinical Microbiology, ASM Press, Washington D.C.; and Shulman et al. (1997) The Biologic and Clinical Basis of Infectious Diseases, W B Saunders, Philadelphia Pa.
In view of the conditions or diseases associated with M. catarrhalis, it would be advantageous to provide specific methods for the diagnosis, prevention, and treatment of diseases attributed to M. catarrhalis. Relevant methods would be based on the expression of M. catarrhalis-derived nucleic acid sequences. Such traits as virulence, acquisition of resistance factors, and effects of treatment using particular therapeutic agents may be characterized by under- or over-expression of nucleic acid sequences as revealed using PCR, hybridization or microarray technologies. Treatment for diseases attributed to M. catarrhalis can then be based on expression of these identified sequences or their expressed proteins, and efficacy of any particular therapy and development of resistance monitored. The information provided herein provides the basis for understanding the pathogenicity of M. catarrhalis and treating and monitoring the treatment of diseases caused by M. catarrhalis. 
The present invention relates to a genomic library comprising the combination of nucleic acid molecules from Moraxella catarrhalis, presented as SEQ ID NOs: 1-41. The library substantially provides the nucleic acid molecules comprising the genome of M. catarrhalis, and the nucleic acid molecules provide a plurality of open reading frames (ORFs). The ORFs uniquely identify structural, functional, and regulatory genes of M. catarrhalis. The invention encompasses oligonucleotides, fragments, and derivatives of the M. catarrhalis nucleic acid molecules, and sequences complementary to the nucleic acid molecules listed in the Sequence Listing.
M. catarrhalis nucleic acid molecules, fragments, derivatives, oligonucleotides, and complementary sequences thereof, can be used as probes to detect, amplify, or quantify M. catarrhalis genes, ORFs, cDNAs, or RNAs in biological, solution or substrate-based, assays or as compositions in diagnostic kits. The invention contemplates the use of such diagnostic probes to identify the presence of M. catarrhalis sequence in a sample or to screen for virulence factors and mutations.
The invention also provides for the comparison of the M. catarrhalis genomic library or the encoded proteins with genomes, individual DNA sequences, or proteins from other Moraxella species or strains, other bacteria, and other organisms to identify virulence factors, regulatory elements, drug targets, and to characterize genomic organization. In another aspect, the present invention provides for the use of computer databases to make such comparisons.
The invention further provides host cells and expression vectors comprising nucleic acid molecules of the invention and methods for the production of the proteins they encode. Such methods include culturing the host cells under conditions for expression of M. catarrhalis protein and recovering the protein from cell culture. The invention still further provides purified M. catarrhalis protein of which at least a portion is encoded by a nucleic acid molecule selected from the nucleic acid molecules of the Sequence Listing.
The subject invention provides a method of screening a library or a plurality of molecules or compounds for specific binding to a M. catarrhalis nucleic acid molecule or fragment thereof or protein or portion thereof, to identify at least one ligand which specifically binds the M. catarrhalis nucleic acid molecule or protein. Such a method comprises the steps of combining the M. catarrhalis nucleic acid molecule or protein with a library or a plurality of molecules or compounds under conditions to allow specific binding and detecting M. catarrhalis nucleic acid molecule or protein bound to at least one molecule or compound, thereby identifying a ligand which specifically binds the nucleic acid molecule or protein. Suitable libraries of ligands comprise aptamers, DNA molecules, RNA molecules, peptide nucleic acids, peptides, mimetics, proteins, agonists, antagonists, antibodies, inhibitors, immunoglobulins, pharmaceutical agents, and drug compounds.
The subject invention also provides a method of purifying a ligand from a sample. Such a method comprises the steps of combining the M. catarrhalis nucleic acid molecule or protein with a library or a plurality of molecules or compounds under conditions to allow specific binding, detecting M. catarrhalis nucleic acid molecule or protein bound to at least one molecule or compound, recovering the bound M. catarrhalis nucleic acid molecule or protein and separating the bound M. catarrhalis nucleic acid molecule or protein from the ligand, thereby obtaining purified ligand.
The invention further comprises an antibody specific for a purified M. catarrhalis protein or a portion thereof which is encoded by an M. catarrhalis nucleic acid molecule selected from the Sequence Listing. Antibodies produced against M. catarrhalis protein may be used diagnostically for the detection of M. catarrhalis proteins in biological, solution- or substrate-based, samples and therapeutically to neutralize the activity of an M. catarrhalis protein expressed during infections caused by M. catarrhalis. 
The Sequence Listing is a compilation of the consensus sequences of contiguous sequences (contigs) or groups of overlapping sequences, assembled from individual sequences obtained by sequencing genomic clone inserts of a randomly generated M. catarrhalis DNA library. Each assembled contig or singlet is identified by a sequence identification number (SEQ ID NO) and by the contig number which it represents.
Table 1 lists the assembled M. catarrhalis contiguous sequences prepared as described in the Examples. The first column contains the number of the contig, which is also SEQ ID NO, listed in ascending order. The second column contains the length of the nucleic acid molecule. The third and fourth columns contain the start and stop nucleotides, respectively, for any open reading frames (ORFs) in the contig. The fifth column contains the Locus ID. The sixth column lists the GenBank identification number of the closest homolog, if any. The seventh column gives the P-value for the match to the homolog. The last column contains the description of the homolog. Orphans or LURs have no GenBank homologs.
Table 2 shows the order of the contigs or singlets comprising the M. catarrhalis genome.
It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms xe2x80x9caxe2x80x9d, xe2x80x9canxe2x80x9d, and xe2x80x9cthexe2x80x9d include plural reference unless the context clearly dictates otherwise. For example, a reference to xe2x80x9ca host cellxe2x80x9d includes a plurality of such host cells known to those skilled in the art.
All patents and publications cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which might be used in connection with the invention are expressly incorporated by reference. Citation is for the purpose of providing the best description of the invention and is not to be construed as an admission that the invention is not entitled to antedate such disclosure.
xe2x80x9cBiologically activexe2x80x9d refers to a protein having structural, immunological, regulatory, or chemical functions of a naturally occurring, recombinant, or synthetic molecule.
xe2x80x9cComplementaryxe2x80x9d refer to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T forms hydrogen bonds with its complements T-G-C-A or U-G-C-A. The degree of complementarity between nucleic acid strands affects the efficiency and strength of the hybridization and amplification reactions.
xe2x80x9cDerivativexe2x80x9d refers to the chemical modification of a nucleic acid or amino acid molecule. Chemical modifications can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process which retains or enhances biological activity, stability, or lifespan of the molecule.
xe2x80x9cFragmentxe2x80x9d refers to an Incyte clone or any part of a nucleic acid molecule which retains a usable, functional characteristic. Useful fragments include oligonucleotides which may be used in hybridization or amplification technologies or to regulate replication, transcription or translation.
xe2x80x9cHybridization complexxe2x80x9d refers to a complex between two nucleic acid molecules by virtue of the formation of hydrogen bonds between purines and pyrimidines.
xe2x80x9cLigandxe2x80x9d refers to any molecule or compound which will bind to a complementary site on a nucleic acid molecule or protein.
xe2x80x9cModulatesxe2x80x9d refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule or compound and either a nucleic acid molecule or a protein.
xe2x80x9cMoleculesxe2x80x9d is used substantially interchangeably with the terms agents and compounds. Such molecules modulate the activity of nucleic acid molecules or proteins of the invention and may be composed of at least one of the following: inorganic and organic substances including cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids.
xe2x80x9cNucleic acid moleculexe2x80x9d is substantially interchangeable with the term polynucleotide and may refer to a probe, a fragment of DNA or RNA of genomic or synthetic origin. Such molecules may be double-stranded or single-stranded and may be engineered into vectors to perform a particular activity such as transcription.
xe2x80x9cOligonucleotidexe2x80x9d is substantially equivalent to the terms xe2x80x9camplimerxe2x80x9d, xe2x80x9cprimerxe2x80x9d, xe2x80x9coligomerxe2x80x9d, and xe2x80x9celementxe2x80x9d, and is preferably single stranded.
xe2x80x9cProteinxe2x80x9d refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
xe2x80x9cPortionxe2x80x9d refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules or compounds which specifically bind to that portion or for the production of antibodies.
xe2x80x9cSamplexe2x80x9d is used in its broadest sense. A sample containing nucleic acid molecules may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a hair, and the like.
xe2x80x9cSubstantially purifiedxe2x80x9d refers to nucleic acid molecules or proteins that are isolated or separated from their natural environment and are about 60% free to about 90% free from other components with which they are naturally associated.
xe2x80x9cSubstratexe2x80x9d refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.